Cloning and Mutagenesis of Catechol 2,3-Dioxygenase Gene from the Gram-Positive <b><i>Planococcus</i></b> sp. Strain S5

Abstract

In this study, the catechol 2,3-dioxygenase gene that encodes a 307- amino-acid protein was cloned from Planococcus sp. S5. The protein was identified to be a member of the superfamily I, subfamily 2A of extradiol dioxygenases. In order to study residues and regions affecting the enzyme's catalytic parameters, the c23o gene was randomly mutated by error-prone PCR. The wild-type enzyme and mutants containing substitutions within either the C-terminal or both domains were functionally produced in Escherichia coli and their activity towards catechol was characterized. The C23OB65 mutant with R296Q substitution showed significant tolerance to acidic pH with an optimum at pH 5.0. In addition, it showed activity more than 1.5 as high as that of the wild type enzyme and its K_m was 2.5 times lower. It also showed altered sensitivity to substrate inhibition. The results indicate that residue at position 296 plays a role in determining pH dependence of the enzyme and its activity. Lower activity toward catechol was shown for mutants C23OB58 and C23OB81. Despite lower activity, these mutants showed higher affinity to catechol and were more sensitive to substrate concentration than nonmutated enzyme.

Activity of a Carboxyl-Terminal Truncated Form of Catechol 2,3-Dioxygenase fromPlanococcussp. S5

“…In order to determine the effect of mutations (C103R, K289Stop) on the structure and activity of catechol 2,3-dioxygenase from Planococcus strain, the 918-bp insert encoding carboxyl-terminal truncated form of C23O in the clone DH5C23OB61 [22] was subcloned into pET-22(b) vector to express the mutated enzyme in E. coli BL21. Localization of mutations was then confirmed by sequence analysis and amino acid sequence of the mutant enzyme was deduced.…”

“…Substitution of arginine at position 296 with glutamine shifted an optimum pH of catechol 2,3-dioxygenase from Planococcus strain from 7.0 to 5.0. Activity of this mutant protein at pH 5.0 and pH 4.0 relative to its activity at pH 7.0 was 144.7% and 124%, respectively [22]. In contrast, mutated C23O from Pseudomonas strain became more alkalescency stable compared with the wild-type enzyme and retained 75% of the maximal enzyme activity even under pH 9.5 [37].…”

“…The structural gene for this enzyme (GenBank ID: HQ223337.2 ) was found in the plasmid and the nucleotide sequencing, and homology search revealed that it shares the greatest homology with isoenzymes from Gram-negative Pseudomonas strains [20]. Additionally, the product analysis and its sequence alignment with other dioxygenases suggest that C23O from strain S5 belongs to the type I superfamily of extradiol dioxygenases [22]. …”

“…From a random library of mutants 8 clones carrying at least one nonsilent mutation were obtained. In one of those mutant form of catechol 2,3-dioxygenase TGC codon for cysteine at position 103 had been changed to a CGC codon for arginine and the AAG codon at position 289 for lysine had been changed to a UAG stop codon [22]. While Cys103Arg amino acid substitution is localized within inactive N-terminal domain, Lys289stop substitution resulting in shortened protein is localized within C-domain of the enzyme [22].…”

“…In one of those mutant form of catechol 2,3-dioxygenase TGC codon for cysteine at position 103 had been changed to a CGC codon for arginine and the AAG codon at position 289 for lysine had been changed to a UAG stop codon [22]. While Cys103Arg amino acid substitution is localized within inactive N-terminal domain, Lys289stop substitution resulting in shortened protein is localized within C-domain of the enzyme [22]. As the C-terminal domain of all known C23Os is catalytically active [17, 23] the aim of the present study was to determine the effect of removal of terminal amino acid residues on catechol 2,3-dioxygenase structure and activity.…”